Generation of Clonal Mesenchymal Progenitors and Mesenchymal Stem Cell Lines Under Serum-Free Conditions

ABSTRACT

Methods for obtaining multipotent Apelin receptor-positive lateral plate mesoderm cells, mesenchymal stem cells, and mesangioblasts under serum-free conditions are disclosed

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part application of U.S. patentapplication Ser. No. 12/554,696, which is a continuation application ofU.S. patent application Ser. No. 12/024,770, which claims the benefit ofU.S. Provisional Patent Application No. 60/974,980, filed Sep. 25, 2007;and U.S. Provisional Patent Application No. 60/989,058, filed Nov. 19,2007, each of which is incorporated herein by reference as if set forthin its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with United States government support awarded bythe following agency: NIH RR052085 and NIH HD044067. The United Statesgovernment has certain rights in this invention.

BACKGROUND

The invention relates generally to clonal primate mesenchymalprogenitors and to mesenchymal stem cell (MSC) lines and methods foridentifying and generating such cells, and more particularly to methodsfor generating clonal mesenchymal progenitors and MSC lines underscrum-free conditions. The invention further relates to a population ofshared endothelial- and mesenchymal cell precursors and methods foridentifying and generating such cells. The invention furthermore relatesto a population of cells comprising lateral plate mesoderm cells andmethods for their generation and isolation from cultured pluripotentstem cells.

During embryonic development of animals, gastrulation forms three germlayers, i.e., endoderm, ectoderm, and mesoderm, that each give rise todistinct bodily cells. Mesoderm develops from primitive streak, atransient embryonic structure formed at the onset of gastrulation.Nascent mesoderm transitionally differentiates into paraxial mesoderm,intermediate mesoderm, and lateral plate mesoderm. Paraxial mesodermgives rise to axial skeleton, and skeletal muscles. Intermediatemesoderm forms the urogenital system. Lateral plate mesoderm gives riseto the circulatory system, including blood cells, vessels, and heart,and forms the viscera and limbs. Extraembryonic mesoderm is locatedoutside the developing embryo. Evidence suggests that extraembryonicmesoderm is derived from the primitive streak during gastrulation(Boucher and Pedersen, Reprod. FertiL Dev. 8:765 (1996)). Extraembryonicmesoderm gives rise to several tissues that provide the embryo withnutrients, a means of waste disposal, and mechanical protection.

Both lateral plate and extraembryonic mesoderm can generate endothelialand blood cells and express FOXF1, HAND1, HAND2, GATA-2, BMP4, andWNT5a, expression of which is low or undetectable in paraxial andintermediate mesoderm (Mahlapuu et al., Development. 128(2):155 (2001);Firulli et al., Nat. Genet. 18(3):266 (1998); Morikawa et al., Circ.Res. 103(12):1422 (2008); Kelley et al., Dev. Biol. 165:193 (1994);Silver et al., Blood 89(4):1154 (1997); Fujiwara et al., Proc. Natl.Acad. Sci. 98(24):13739 (2001); Takada et al., Genes Dev. 8(2):174(1994)). Distinctive markers for lateral plate and extraembryonicmesoderm remain to be elucidated. A recent finding by Bosse et al.suggests that IRX3 is expressed in lateral plate mesoderm but not inextraembryonic mesoderm (Bosse et al., Mech. Dev. 69(1-2):169 (1997)).For the purposes of this application, the term lateral plate is used todescribe both tissues.

Certain committed mesodermal progenitors can give rise to cells of morethan one lineage. Example of such progenitors includes hemangioblasts,which can give rise to both hematopoietic- and endothelial cells. ChoiK, et al., “A common precursor of hematopoietic and endothelial cells,”Development 125:725 (1998).

MSCs can differentiate into at least three downstream mesenchymal celllineages (i.e., osteoblasts, chondroblasts, and adipocytes). To date, nounique MSC marker has been identified. As such, morphological andfunctional criteria are used to identify these cells. See, Horwitz E, etal., “Clarification of the nomenclature for MSC: the InternationalSociety for Cellular Therapy position statement,” Cytotherapy 7:393(2005); and Dominici M, et al., “Minimal criteria for definingmultipotent mesenchymal stromal cells. The International Society forCellular Therapy position statement,” Cytotherapy 8:315 (2006). BecauseMSCs can differentiate into many cell types, the art contemplatesmethods for differentiating MSCs for cell-based therapies, forregenerative medicine and for reconstructive medicine.

Typically, MSCs are isolated from adult bone marrow, fat, cartilage andmuscle. Pittenger F, et al., “Multilineage potential of adult humanmesenchymal stem cells,” Science 284:143-147 (1999); Zuk P, et al.,“Multilineage cells from human adipose tissue: implications forcell-based therapies,” Tissue Eng. 7:211-228 (2001); and Young H. etal., “Human reserve pluripotent mesenchymal stem cells are present inthe connective tissues of skeletal muscle and dermis derived from fetal,adult, and geriatric donors,” Anat. Rec. 264:51-62 (2001). MSCs havealso been isolated from human peripheral blood. Kassis I, et al.,“Isolation of mesenchymal stem cells from G-CSF-mobilized humanperipheral blood using fibrin microbeads,” Bone Marrow Transplant.37:967-976 (2006). MSCs can also be isolated from human neonatal tissue,such as Wharton's jelly (Wang H, et al., “Mesenchymal stem cells in theWharton's jelly of the human umbilical cord,” Stem Cells 22:1330-1337(2004)), human placenta (Fukuchi Y, et al., “Human placenta-derivedcells have mesenchymal stem/progenitor cell potential,” Stem Cells22:649-658 (2004)); and umbilical cord blood (Erices A, et al.,“Mesenchymal progenitor cells in human umbilical cord blood,” Br. J.Haematol. 109:235-242 (2000)) and human fetal tissues (Campagnoli C, etal., “Identification of mesenchymal stem/progenitor cells in humanfirst-trimester fetal blood, liver, and bone marrow,” Blood 98:2396-2402(2001)).

The art is limited by an inability to isolate sufficient MSCs forsubsequent differentiation and use. Where suitable donors are available,the invasive procedures required to isolate even a limited number ofcells present risks to donors. It also remains difficult to maintainisolated MSCs in long-term culture and to maintain such cultures free ofbacterial or viral contamination.

Efforts to devise methods for differentiating embryonic stem cells(ESCs) including human ESCs (hESCs) to MSCs either have requiredculturing the cells in a medium containing potentially contaminatingserum or have yielded cells that retain characteristics ofundifferentiated hESCs. For example, Barberi et al. differentiated hESCsto MSCs on mitotically-inactivated mouse stromal cell lines (i. e.;feeder cells) with 20% heat-inactivated fetal bovine serum (FBS) inalpha MEM medium for 40 days. Barberi T, et al. “Derivation ofmultipotent mesenchymal precursors from human embryonic stern cells,”PLoS Med. 2:el61 (2005). Cells were harvested and assayed for CD73, andCD73+ cells were then plated in the absence of the feeder cells with 20%FBS in alpha MEM for 7 to 10 days. Barberi et al. differentiated theMSCs into adipogenic cells, chondrogenic cells, osteogenic cells andmyogenic cells.

Likewise, Olivier et al. differentiated hESCs to MSCs by platingraclures (i.e., spontaneously differentiated cells that appear in hESCculture in the center or at the edges of colonies) with Dl0 medium(DMEM, 10% FBS, 1% penicillin/streptomycin and 1% non-essential aminoacids) changed weekly until a thick, multi-layer epithelium developed.Olivier E, et al., “Differentiation of human embryonic stem cells intobipotent mesenchymal stem cells,” Stem Cells 24:1914-1922 (2006). Afterapproximately four weeks, MSCs were isolated by dissociating theepithelium with a mixture of trypsin, collagenase type IV and dispasefor four to six hours, followed by re-plating in D10 medium. Olivier etal.'s MSCs grew robustly, had stable karyotypes, were contact inhibited,senesced after twenty passages and differentiated into adipogenic andosteogenic cells. Olivier et al. did not report that the cellsdifferentiated into chondroblasts. Unlike Barberi et al., Olivier et al.did not require feeder cells to support differentiation of hESC to MSCs.However, Olivier et al's MSCs were SSEA-4 positive, suggesting thatthese MSCs expressed cell surface markers characteristic of hESC.

Pike & Shevde differentiated hESCs to MSCs via embryoid bodies (EBs)incubated for ten to twelve days in a mesenchymal-specific medium(MesenCult® medium with 10% FBS; alpha MEM with glutamine andnucleosides; or DMEM with glucose and glutamine, replaced every twodays). US Patent Publication No. 2006/0008902. The EBs were digested,and pre-mesenchymal cells were cultured to 80% confluence. The cellswere trypsinized and passaged three times in mesenchymal-specificmedium.

Meuleman et al. reported culturing MSCs in a serum-free medium; however,it was later discovered that the medium did in fact contain animal serumas a component. Meuleman N, et al., “Human marrow mesenchymal stem cellculture: serum-free medium allows better expansion than classicalalpha-minimal essential medium (MEM),” Eur. J. Haematol. 76:309-316(2006); and Meuleman N, et al., “Human marrow mesenchymal stem cellculture: serum-free medium allows better expansion than classicalalpha-minimal essential medium (MEM),” Eur. J. Haematol. 77:168 (2007);but see, Korhonen M, “Culture of human mesenchymal stem cells inserum-free conditions: no breakthroughs yet,” Eur. J. Haematol. 77:167(2007).

Those methods cultured and differentiated MSCs in serum-containingmedium. Serum-free conditions for culturing and differentiating MSCs, ifdefined, would reduce variation among batches and eliminate a risk ofinfection transmitted by xenogenic by-products and pathogens.Sotiropoulou P, et al., “Cell culture medium composition andtranslational adult bone marrow-derived stem cell research,” Stem Cells24:1409-1410 (2006).

For the foregoing reasons, there is a need for new methods for obtainingearly mesenchymal progenitors and MSCs, especially when derived underserum-free conditions.

Mesoderm and the neural crest can both give rise to mesenchymalprecursors during embryonic development. Dennis, J. E., and P. Charbord,“Origin and differentiation of human and murine stroma,” Stem Cells20:205-214 (2002); Takashima, Y, et al., ”Neuroepithelial cells supplyan initial transient wave of MSC differentiation,” Cell 129:1377-1388(2007). While conditions for generating MSCs of neural crest origin fromembryonic stem cells have been described, Takashima et al., supra; Lee,G, et al., “Isolation and directed differentiation of neural crest stemcells derived from human embryonic stem cells,” Nat Biotechnol25:1468-1475 (2007), it is not known how to generate MSCs from mesoderm.

For the foregoing reasons, there is a need for new methods for obtainingearly mesenchymal progenitors and MSCs, particularly under serum-freeconditions. Further, there is a need to identify and generatemesoderm-derived MSCs as well as early mesodermal progenitors that cangive rise to MSCs during differentiation of pluripotent stem cells intoMSCs.

BRIEF SUMMARY

The invention generally relates to a newly identified common mesenchymaland endothelial cell precursor, i.e., mesangioblasts, derived from invitro-differentiated stem cells.

In a first aspect, the invention is summarized in that a method ofgenerating a clonal population of primate MSCs includes the steps ofculturing a heterogeneous, single-cell suspension of primate cells thatcontains mesenchymal progenitors in a serum-free, semi-solid mediumcontaining between about 5 and about 100 ng/ml bFGF until independentcolonies form, and culturing one of the independent colonies in aserum-free, liquid medium containing between about 5 and about 100ng/ml, or at about 5 ng/ml, or between about 20 and about 100 ng/ml,bFGF to obtain an substantially pure clonal population of MSCs.

The heterogeneous suspension for use in the method can be obtained, forexample, by differentiating pluripotent cells from a primate (e.g.,human), such as ESCs or induced pluripotent stem (iPS) cells, in cultureuntil cells in the culture are mesenchymal progenitors. This can beaccomplished by co-cuilturing the pluripotent cells with bone marrowstromal cells in a medium that supports differentiation as describedherein for at least two to five days, or by dissociating EBs, which canthemselves be obtained by culture of pluripotent cells using well-knownmethods, and then suspending the cells as a single cell suspension, Thebone marrow stromal cells can be mouse OP9 cells. A heterogeneoussuspension substantially free of some or all cells not derived by invitro differentiation of pluripotent cells (especially co-cultured bonemarrow cells) can be obtained by depleting those cells from thesuspension. These cells can be depleted from the suspension before use,for example, by non-covalently binding the cells to be depleted toparamagnetic monoclonal antibodies specific for the epitopes on thecells to be depleted and then segregating the antibody-bound cells witha magnet. Cells in a suspension obtained from pluripotent cells canexpress at least MIXL1 and T (BRACHYURY).

The medium can be rendered semi-solid by including about 1%methylcellulose in the medium. The medium can optionally contain betweenabout 10 and about 20 ng/ml PDGF-BB. The suspension can be cultured forbetween about ten to about twenty days or more to produce the colonies.

Mesenchymal progenitors are identified as having been present in thesuspension if mesenchymal colonies form during culture in theserum-free, semi-solid medium supplemented with bFGF. An example of suchbFGF-dependent colony-forming assay for detecting mesenchymal progenitoris described in U.S. Pat. No. 7,615,374, incorporated herein as if setforth in its entirety. The colonies obtained in the colony-forming assaycan be identified as mesenchymal by their expression of at least aplurality of FOXF1, MSX1, MSX2, SNAI1, SNAI2, SOX9 and RUNX2.Characteristics of the colonies include functional, morphological andphenotypical characteristics and gene expression profile. Functionalcharacteristics of the colonies include (1) growth stimulation byfactors that promote mesenchymal cell growth (e.g., PDGF-BB, EGF andTGF-alpha) and growth suppression by factors involved in mesodermaldifferentiation (e.g., VEGF, TGF-beta and Activin A); (2)differentiation into osteogenic, chondrogenic or adipogenic celllineages; and (3) differentiation into endothelial cells. Morphologicalcharacteristics of the colonies include (1) tight packing of cells toform round (i.e., spherical) aggregates measuring 100-500 μm indiameter; (2) colony formation through establishing tightly packedstructures (cores) that further develop into compact spheroid colonies;and (3) even after prolonged culture, lack of dense outer cell layer andirregular inner structure, which are characteristics of EBs.Phenotypical characteristics of the colonies include (1) expression ofCD44, CD56, CD105 and CD140a (PDGFRA), CD146, but not hematoendothelialsurface markers (i.e., CD31, CD43, CD45 and VE-cadherin); (2) expressionof FOXF1, MSX1, MSX2, SNAI1, SNAI2, SOX9 and RUNX2; and (3) expressionof vimentin, alpha smooth muscle actin, and desmin.

The mesenchymal colonies thus formed in the method can be furthercultured in the presence of an extracellular matrix protein, such asMatrigel®, collagen, gelatin or fibronectin, as well as combinationsthereof.

The invention is further summarized as a substantially pure populationof clonally-derived MSC lines produced from the methods described abovethat are positive for at least CD44, CD56, CD 73, CD105, CDl40a, andCD146, but negative for CD31, CD43, CD45 and VE-cadherin.

The described embodiments have many advantages, including thatmesenchymal progenitors and MSCs obtained in the methods may be used totreat diseases associated with bone, cartilage and fat cells.

It is also an advantage that a clonal population of MSCs can be obtainedfrom a single mesenchymal colony.

It is also an advantage that the cells obtained in the methods caneasily be selected for further expansion because the mesenchymalprogenitors have high proliferation potential and form large colonies.

It is yet another advantage that cells obtained in the methods can betolerant or tolerogenic to allo- and auto-immune response ontransplantation.

It is still another advantage that the cells obtained in the methods candifferentiate into at least osteogenic, chondrogenic and adipogeniclineages.

It is still another advantage that mesenchymal colonies obtained in themethods possess angiogenic potential.

The invention is further summarized as a population of in vitro-derivedApelin receptor-positive (APLNR⁺) lateral plate mesoderm cells. Thesecells can be isolated from mixed populations of differentiatingpluripotent stem cells based on expression of the Apelin receptor(APLNR). These cells can differentiate into cells of the body wall andviscera and give rise to mesangiogenic mesenchymal and hemangiogenicblast colonies in semisolid media cultures in the presence of bFGF. TheAPLNR⁺ cells express transcripts characteristic of mesoderm,specifically lateral plate mesoderm.

It is still another advantage of the invention that MSCs obtained by theclaimed methods are of mesodermal origin and can be derived from APLNR⁺cells enriched in lateral plate mesoderm cells.

These and other features, aspects and advantages of the presentinvention will become better understood from the description thatfollows. The description of preferred embodiments is not intended tolimit the invention to cover all modifications, equivalents andalternatives. Reference should therefore be made to the claims hereinfor interpreting the scope of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-E illustrate the properties of two types of hESC-dcrivedcolonies, i.e., mesenchymal colonies derived from mesangioblasts (MB)and blast colonies derived from hemangioblasts (HB). FIG. 1A depicts MBand HB colony morphologies following growth in semisolid media for 3, 5,7, and 12 days. FIG. 1B depicts the kinetics of FGF-dependent colonyformation. Bars represent standard deviation of four independentexperiments. Depending on whether the hESC-derived single cells areinitially co-cultured with OP9 cells for 2 days or 3 days, they assumeeither MB or HB potential. FIG. 1C illustrates that bFGF, but not PDGFor VEGF alone, supports both MB and HB colony formation. The data arerepresented as mean±SD(n=4). The asterisk indicates statisticalsignificance (p<0.01) between cultures containing FGF alone and FGF incombination with either PDGF or VEGF. FIG. 1D illustrates thedifferentiation potential of MB-derived and HB-derived colonies aftercoculture with OP9 cells for 4 days. Flow cytometry demonstrated that MBcolony-derived cells collected on day 12 of clonogenic culture gave riseto CD146⁺CD31⁻ mesenchymal and CD3l ⁺CD43⁻ endothelial cells, while HBcolony-derived cells gave rise to CD31⁺CD43⁻ endothelial cells and CD43⁺hematopoietic lineage cells. FIG. 1E illustrates immunostaining analysisof cell clusters developed from a single MB (top, scale bar, 100 μm) andHB (bottom, scale bar, 50 μm) colony collected on day 5 of clonogenicculture. Cells were identified as CD144⁺ (also known as VE-cadherin)CD43⁻ endothelial, CD43⁺ hematopoietic, and calponin⁺CD144⁻ mesenchymal.The scale bars represent 100 μm. Colonies developed from cell clustersof a single MB colony generate calponin⁺CD144(VE-cadherin)⁻mesenchymalcells and CD144(VE-cadherin)⁺calponin⁻endothelial cells (upper panel).Colonies developed from cell clusters developed from a single HB colonygenerate CD43⁺ hematopoietic and CD144(VE-cadherin)⁺CD43⁻ endothelialcells (lower panel).

FIG. 2 illustrates microarray analysis of gene expression in hESCsco-cultured with OP9 cells from day 0 (H1) to day 7. FIG. 2A depicts aheat map for selected gene sets defining particular germ layers andtheir subpopulations and derivatives. FIG. 2B depicts relative geneexpression of MIXL1, T, SNAI1, FOXF1, and SOX17 as determined byquantitative PCR. FIG. 2C depicts the fold increase in the number ofhESC-derived cells after day 1-6 of OP9 co-culture in relation toprevious day. The data is represented as means±SD (n=3).

FIG. 3 illustrates analysis of APLNR⁺cells. FIG. 3A depicts dot plots offlow cytometry results. FIG. 3B depicts the effect of inhibitors ofmesoderm formation (SB431542 (5 μg/ml) and DKK1 (150 μg/ml)) ongeneration of APLNR+cells from H1 cells in OP9 cell co-cultures. FIG. 3Ccompares transcript expression between APLNR+ and APLNR⁻ cells. FIG. 3Ddepicts the colony-forming potential of APLNR⁺ and APLNR⁻ cells.

FIG. 4 illustrates the gene expression profiles of APLNR⁺ cells, APLNR⁻cells, cores, colonies, and a mesenchymal stem cell (MSC) line (atpassages p1 and p5) obtained from H1 hESCs differentiated for 2 (D2) or3 (D3) days by coculture with OP9 cells. FIG. 4A depicts heat maps forselected sets of genes defining indicated germ layers and theirsubpopulations/derivatives. Cores were collected on day 3 of clonogeniccultures and fully developed colonies on day 12 of clonogenic cultures.EMT is epithelial-mesenchymal transition. VSMC is vascular smooth musclecells. FIG. 4B shows lack of SOX1 neuroepithelium marker expressionthroughout all stages of differentiation. Embryoid bodies derived fromH1 hESCs differentiated for 14 days were used as positive control. FIG.4C illustrates quantitative RT-PCR analysis of representativetranscripts in indicated cell subsets. Bars represent gene expression inpooled samples from 3 experiments normalized to RPL13.

FIG.5 depicts a schematic diagram of mesodermal lineages development anddifferentiation toward MSCs from pluripotent stem cells.

FIG.6 depicts a schematic diagram of the protocol used for hESCdifferentiation, generation of MB colonies, and clonal MSC lines.

FIG.7 depicts a schematic diagram of the protocol used to evaluatedifferentiation potential of mesenchymal and blast colonies.

DESCRIPTION OF EXEMPLARY EMBODIMENTS

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the invention belongs. Although any methods andmaterials similar to or equivalent to those described herein can be usedin the practice or testing of the present invention, the preferredmethods and materials are described herein. It is commonly understood byone of ordinary skill in the art that “lack of expression” of a gene orthe absence of a certain marker on a cell refers to an inability todetect such gene or marker expression using methods known in the art atthe time of filing. It cannot be ruled out that more sensitive methodscould detect low levels of expression of such genes or markers.

In describing the embodiments and claiming the invention, the followingterminology is used in accordance with the definitions set out below.

As used herein, “about” means within 5% of a stated concentration.

As used herein, “clonal” means a population of cells cultured from asingle cell, not from an aggregate of cells. Cells in a “clonalpopulation” display a substantially uniform pattern of cell surfacemarkers and morphology and are substantially genetically identical.

As used herein, an “embryoid body” or an “EB,” is an aggregate of cellsderived from pluripotent cells, such as ESCs or iPS cells, where cellaggregation can be initiated by hanging drop, by plating upon non-tissueculture-treated plates or spinner flasks (i.e., low attachmentconditions); and any method that prevents the cells from adhering to asurface to form typical colony growth. EBs appear as rounded collectionsof cells and contain cell types derived from all three germ layers (ie.,the ectoderm, mesoderm and endoderm). Methods for generating EBs arewell-known to one having ordinary skill in the art. See, Itskovitz-EldorJ, et al., “Differentiation of human embryonic stem cells into embryoidbodies compromising the three embryonic germ layers,” Mol. Med. 6:88-95(2000); Odorico J, et al., Stem Cells 19:193-204 (2001); and U.S. Pat.No. 6,602,711, each of which is incorporated herein by reference as ifset forth in its entirety.

As used herein, “serum-free” means that neither the culture nor theculture medium contains serum or plasma, although purified or syntheticserum or plasma components (e.g., FGFs) can be provided in the culturein reproducible amounts as described below.

As used here, a “substantially pure population” means a population ofderived cells that contains at least 99% of the desired cell type. Cellpurification can be accomplished by any means known to one of ordinaryskill in the art. For example, a substantially pure population of cellscan be achieved by growth of cells or by selection from a less purepopulation, as described herein.

As used herein, “pluripotent cells” means a population of cells capableof differentiating into all three germ layers and becoming any cell typein the body. Pluripotent cells express a variety of cell surfacemarkers, have a cell morphology characteristic of undifferentiated cellsand form teratomas when introduced into an immunocompromised animal,such as a SCID mouse. Teratomas typically contain cells or tissuescharacteristic of all three germ layers.

As used herein, “multipotent” cells are more differentiated thanpluripotent cells, but are not permanently committed to a specific celltype. Pluripotent cells therefore have a higher potency than multipotentcells.

As used herein, “induced pluripotent stem cells” or “iPS cells” arecells that are differentiated, somatic cells reprogrammed topluripotency. The cells are substantially genetically identical to theirrespective differentiated somatic cell of origin and displaycharacteristics similar to higher potency cells, such as ES cells. See,Yu J, et al., “Induced pluripotent stem cell lines derived from humansomatic cells,” Science 318:1917-1920 (2007), incorporated herein byreference as if set forth in its entirety.

As used herein, a “mesenchymal stem cell” (MSC) is a cell capable ofdifferentiating into the skeletal cell lineages (ie., osteoblasts,chondroblasts and adipocytes). As noted above, no unique MSC marker hasbeen identified. As such, morphological and functional criteriawell-known to those of ordinary skill in the art are used to identifythese cells. See, Horwitz et al., supra; Dominici et al., supra; TrivediP & Hematti P, “Derivation and immunological characterization ofmesenchymal stromal cells from human embryonic stem cells,” Exp.Hematol. Jan. 5, 2008 [Epub ahead of print]; Trivedi P & Hematti P,“Simultaneous generation of CD34+ primitive hematopoietic cells andCD56+ mesenchymal stem cells from human embryonic stem cells coculturedwith murine OP9 stromal cells, “Exp. Hematol. 35:146-154 (2007); and USPublished Patent Application No. 2006/0008902, each of which isincorporated herein by reference as if set forth in its entirety. MSCsproduced by the methods described herein can be characterized accordingto phenotypic criteria. For example, MSCs can be recognized by theircharacteristic mononuclear ovoid, stellate shape or spindle shape, witha round to oval nucleus. The oval elongate nuclei typically haveprominent nucleoli and a mix of hetero- and euchromatin. These cellshave little cytoplasm, but many thin processes that appear to extendfrom the nucleus. It is believed that MSCs will typically stain for one,two, three or more of the following markers: CD106 (VCAM), CD73, CD146,CD166 (ALCAM), CD29, CD44 and alkaline phosphatase, while being negativefor hematopoietic lineage cell markers (e.g., CD14 or CD45) andendothelial lineage cell markers (e.g., CD31 and VE-cadherin). MSCs mayalso express STRO-1 as a marker.

As used herein, a “mesangioblast” is a progenitor for MSCs as well asendothelial cells.

As used herein, a “mesenchymal colony” is a colony composed ofmesenchymal cells originating from mesangioblasts.

As used herein, a “hemangioblast” is a precursor to blood cells as wellas endothelial cells.

As used herein, a “blast colony” is a colony composed of predominantlyhematopoietic cells originating from hemangioblasts.

As used herein, “mesendoderm” is a tissue that gives rise to mesodermand endoderm.

As used herein, “mesoderm” is a cell subset that expresses KDR andPDGFRa to much greater level than POU4F1, SOX1, and PAX6 (neural crestand neuroectoderm), LAMA3, KRT14, and KRT10 (surface ectoderm), CGA andPLAC1 (trophectoderm) FOXA1, FOXA2, APOA1, TMPRSS2, TTR1, and AFP(endoderm), and SOX2 and DPPA2 (undifferentiated hESCs).

As used herein, “lateral plate mesoderm” is a subset of mesoderm thatexpresses at least FOXF1 and HAND1 but lacks expression of MEOX1 andTCF15 (paraxial mesoderm), PAX2 and PAX8 (intermediate mesoderm), and iscapable of at least endothelial and hematopoietic differentiation.

It is contemplated that Matrigel®, laminin, collagen (especiallycollagen type I), fibronectin and glycosaminoglycans may all be suitableas an extracellular matrix, by themselves or in various combinations.

The invention will be more fully understood upon consideration of thefollowing non-limiting Examples.

EXAMPLES Example 1 Generation of MSCs from Pluripotent Stem Cells UnderSerum-Free Conditions

hESCs (H1; WiCell; Madison, Wis.) were maintained on irradiated mouseembryonic fibroblasts in a serum-free medium, such as DMEM/F12 mediumsupplemented with _20% Knockout™ serum replacer, 2 mM L-glutamine,1×(100 μM) non-essential amino acids, 100 μM 2-mercaptoethanol and 4ng/ml bFGF (all from Gibco-Invitrogen; Carlsbad, Calif.). See Amit M, etal., “Clonally derived human embryonic stem cell lines maintainpluripotency and proliferative potential for prolonged periods ofculture,” Dev. Biol. 227:271-278 (2000), incorporated herein byreference as if set forth in its entirety. Mouse OP9 bone marrow stromalcells (kindly provided by Dr. Toru Nakano and available from ATCC,catalog # CRL-2749) were maintained by four-day subculture ongelatin-coated dishes in alpha MEM medium (Gibco-Invitrogen) with 20%fetal calf serum (FCS; HyClone; Logan, Utah).

The hESCs were induced to differentiate by co-culture with mouse OP9bone marrow stromal cells, as previously described. Vodyanik M, et al.,“Human embryonic stem cell-derived CD34+ cells: efficient production inthe coculture with OP9 stromal cells and analysis of lymphohematopoieticpotential, “Blood 105:617-626 (2005), incorporated herein by referenceas if set forth in its entirety. Briefly, small aggregates of hESCs wereadded to OP9 cells in alpha MEM supplemented with 10% FCS and 100 μM MTG(Sigma; St. Louis, Mo.). On the next day (day 1) of culture, the mediumwas changed, and the cultures were harvested on the days indicatedbelow.

On day two of hESC (H1) co-culture with OP9 stromal cells, peakexpression of transcription factors for primitive streak population(mesendoderm) (GSC, EOMES, MIXL1 and T (BRACHYURY)) and early mesoderm(EVX1, LHX1 and TBX6) were detected with NimbleGen® (Madison, Wis.)microarrays.

On days 3-5 of co-culture, the culture contained mesenchymalprogenitors, as well as cells expressing genes characteristic ofendoderm and mesoderm. Among the genes characteristic for mesoderm, onlygenes characteristic of the lateral plate mesoderm, such as FOXF1,HAND1, NKX2-5, and GATA2 were expressed consistently, In contrast, genescharacteristic for the axial (CHRD, SHH), paraxial (MEOX1, TCF15), orintermediate (PAX2, PAX8) mesoderm were not expressed consistently.Thus, hESCs co-cultured with OP9 cells for 3-5 days gave rise to cellsexpressing genes characteristic of the lateral plate/extraembryonicmesoderm. On days 3-5 of hESC (H1)/0P9 co-culture, the cells were alsocharacterized by maximal cell proliferation and sustained expression ofgenes involved in epithelial-mesenchymal transition (EMT, SNAI1, andSNAI2) and cell expansion (HOXB2, HOXB3).

On days 5-7 of hESC (H1)/OP9 co-culture, differentiation into specificmesodermal and endodermal lineages was observed, when markers ofdeveloping endoderm (AFP and SERPINA1), mesenchymal (SOX9, RUNX2, andPPARG2), and hematoendothelial (CDH5 and GATA1) cells were detected.Neither muscle-inductive factors (MYOD1, MYF5, and MYF6) norneuroectoderm (SOX1, PAX6, and NEFL) or trophectoderm (CGB and PLAC)markers were expressed throughout the seven days of co-culture,indicating that OP9 cells provided an efficient inductive environmentfor directed hESC differentiation toward the mesendodermal pathway.

On day 2 of hESC (H1)/OP9 co-culture, a single-cell suspension washarvested from the co-culture by successive enzymatic treatment withcollagenase IV (Gibco-invitrogen) at 1 mg/ml in DMEM/Fl2 medium for 15minutes at 37° C. and 0.05% Trypsin-0.5 mM EDTA (Gibco-Invitrogen) for10 minutes at 37° C. Cells were washed 3 times with PBS-5% FBS, filteredthrough 70 μM and 30 μM cell strainers (BD Labware; Bedford, Mass.) andlabeled with anti-mouse CD29-PE (AbD Serotec; Raleigh, N.C.) and anti-PEparamagnetic monoclonal antibodies (Miltenyi Biotec; Auburn, Calif.).The cell suspension was purified with magnet-activated cell sorting(MACS) by passing it through a LD magnetic column attached to aMidi-MACS separation unit (Miltenyi Biotech) to obtain a negativefraction of OP9-depleted, hESC-derived cells. Purity was verified usingpan anti-human TRA-1-85 monoclonal antibodies (R&D Systems; Minneapolis,Minn.).

The purified single-cell suspension was plated at density of 0.5-2×10⁴cells/ml on a semisolid, serum-free medium composed of StemLine™serum-free medium (Sigma; St. Louis, Mo.) supplemented with 5-100 ng/mlbFGF (PeproTech; Rocky Hill, N.J.) and 1% methylcellulose (Stem CellTechnologies; Vancouver, Canada) with or without 10-20 ng/ml PDGF-BB(PeproTech). PDGF-BB improved growth of mesenchymal cells, but was notessential for colony formation. Alternatively, single cell suspensionswere plated in a semisolid colony-forming serum-free medium containing40% ES-Cult M3120 methylcellulose, 25% serum-free expansion medium(SFEM, Stem Cell Technologies), 25% endothelial serum-free medium(E-SFM, Invotrogen), 10% BIT 9500 (Stem Cell Technologies), GlutaMAX(diluted 1:100), Ex-Cyte (diluted 1:1000, Millipore), 100 μMmonothioglycerol (MTG), 50 μg/ml ascorbic acid and 20 ng/ml bFGF.

After 10-20 days of culture, large, compact mesenchynal colonies formedthat resembled embryoid bodies (EBs). While these mesenchymal colonieswere detected as early as day 7, 10-20 days of culture were required toreveal actively growing colonies. Undifferentiated hESCs or cellsharvested on day 1 or on day 6 of co-culture did not form thesemesenchymal colonies when cultured under the same conditions.

Mesenchymal colonies, which resembled embryoid-like bodies, weredistinguished from EBs through several characteristics: (1) formationand growth under serum-free conditions supplemented with bFGF andstimulation by factors promoting mesenchymal cell growth (eg., PDGF-BB,EGF and TGE-α), but suppression by factors involved in mesodermaldifferentiation (e.g., VEGF, TGF-β and Activin A) in mesenchymalcolonies; (2) lack of a dense outer cell layer and irregular cavitatedstructure characteristic of EBs, even after prolonged culture inmesenchymal colonies; (3) presence of morphological homogeneity in cellscomprising the mesenchymal colonies; and (4) formation of coloniesthrough establishment of tightly packed structures (cores) which furtherdevelop into compact spheroid colonies.

To demonstrate that the single-cell suspensions did not form aggregatesupon plating in semi-solid medium, clonality of the mesenchymal coloniesobtained in the culture methods was tested and confirmed using chimerichESC lines established from cells retrovirally marked with a reportergene, e.g., either enhanced green fluorescent protein (EGFP) or histone2B-(H2BB) mOrange fluorescent protein. Expression of a product of thereporter gene indicated clonality. The chimeric hESC lines weregenerated from two lentiviral constructs: (1) the EGFP protein expressedconstitutively from an elongation factor 1 alpha (EF1alpha) promoter,and (2) the H2BB-mOrange protein expressed constitutively from theEF1alpha promoter. Both constructs were packaged in 293FT cells, and thelentiviruses were used to transduce H1 hESCs to produce stable H1 hESClines that expressed either green EGFP protein or orange H2BB-mOrangeprotein. Mesenchymal colonies derived from the described methods were ofsingle colors, either green or orange, thus indicating the clonal (i.e.,single cell) origin of the MSCs. In addition, prospective phenotypicanalysis demonstrated a positive correlation between mesenchymal-colonyforming cell (CFC) frequency and KDR (VEGFR2) expression, thoughKDR^(high)CD34+ population of the earliest hemangiogenic precursors wasdevoid of mesenchymal-CFCs. Analysis of cells within mesenchymalcolonies revealed a homogeneous population of early mesenchymal cellsdefined by high, CD90, CD140a and CD166 expression, low CD44, CD56 andCD105 expression and lack of CD24, CD31, CD43, CD45, CD144(VE-cadherin), and lack of SSEA4 expression. In addition, mesenchymalcolonies expressed vimentin, alpha smooth muscle actin, and desmin.Furthermore, mesenchymal colonies expressed genes specific for MSClineage, such as FOXF1, MSX1, MSX2, SNAI1, SNAI2, SOX9, and RUNX2.

Individual mesenchymal colonies were transferred to wells of a collagen-or fibronectin-coated, 96-well plate pre-filled with 0.2 ml/wellStemLine™ serum-free medium supplemented with 5-100 ng/ml bFGF orserum-free expansion medium consisting of 50% StemLine II serum-free HSCexpansion medium (H-SFEM, Sigma), and 50% E-SFM supplemented withGlutaMAX (diluted 1:100), ExCyte (diluted 1:2000), 100 μM MTG, and 10ng/ml bFGF. After 3-4 days of culture, adherent cells from individualwells were harvested by trypsin treatment and expanded on collagen- orfibronectin-coated dishes in StemLine™ serum-free medium with 5-100ng/ml bFGF or serum-free expansion medium (M-SFEM) containing 50%StemnLine™ II serum-free HSC expansion medium (HSFEM; Sigma), 50% E-SFM,GlutaMAX™ (1/100 dilution), Ex-Cyte® supplement (1/2000 dilution), 100μM MTG, and 5-100 ng of bFGF.

MSCs were expanded for many passages. When individual colonies wereplated on collagen- or fibronectin-coated plates, immediate attachmentand vigorous outgrowth of fibroblast-like cells were observed. Duringsubsequent passages, cells grew intensively during the first 10passages; however, growth rate was attenuated at passages 10-15 andgradual senescence was observed during passages 15-20. Cultures derivedfrom single MB-CFC accumulated up to 10²² total cells in the observedtime period. Because each colony is presumed to have originated from asingle cell, the number corresponds to the expansion potential of asingle hESC-derived mesenchymal precursor.

Cell lines established from individual colonies were maintained inserum-free medium with bFGF for 10-15 passages at a high proliferationrate. All cell lines displayed a mesenchymal phenotype, characterized byexpression of CD44, CD56, CD 73, CD105, CD146, and CD140a (PDGFRA) andlack of hematoendothelial markers (i.e. CD31, CD43, CD45 andVE-cadherin). When tested in conditions revealing mesenchymaldifferentiation potential, the cell lines were capable of osteogenic,chondrogenic and adipogenic differentiation. Interestingly, these cellsresemble bone marrow MSCs, but expand and proliferate better than bonemarrow MSCs. These expanded mesenchymal cells could be differentiatedinto cells of the chondro-, osteo- and adipogenic lineage. However,these cells could not give rise to hematopoietic or endothelial cellswhen cultured with OP9 cells, or when cultured in feeder-free cultureswith hematoendothelial growth factors (VEGF, bFGF, SCF, TPO, IL3, IL6),indicating a limited differentiation potential of these mesenchymalcells.

Mesenchymal colonies were also generated from various inducedpluripotent stem (iPS) cells, such as iPS(IMR90)-1, iPS(SK)-46, and iPS(FSK)-1 reprogrammed using a lentiviral vector (Yu et al., Science318:1917-1920 (2007)), or transgene-free iPS-5 4-3-7T and iPS-1 19-9-7T(Yu et al., Science 324:797-801 (2009)). Mesenchymal colonies derivedfrom transgene-containing iPS cells displayed irregular or more loosemorphology. Transgene-free iPSC produced typical spheroid mesenchymalcolonies.

Example 2 In Vitro Generation and Characterization of Mesangioblasts

To isolate and characterize a population of mesodermal progenitors thatcan give rise to cells of the mesodermal lineage with hematopoietic,endothelial, and mesenchymal stem cell potentials, H1 hES cells wereco-cultured with OP9 cells, as described in Example 1. After two orthree days of co-culture, when genes representative of primitive streakpopulation (mesendoderm) (MIXL1, T, EOMES) were expressed, thehESC-derived cells depleted of 0P9 cells using anti-mouse CD29 antibodywere plated in semisolid, serum-free medium, essentially as described inExample 1, with 20 ng/ml bFGF (Pepro Tech; Rocky Hill, N.J.). The numberof colony-forming cells (CFCs) was calculated per 1000 plated H1-derivedTRA-1-85⁺ cells.

After 2-3 days in semisolid medium, the cells formed tightly packedstructures (cores). Cores derived from hESCs that were differentiated inco-culture with OP9 cells for 2 days further grew into spheroidmesenchymal colonies. Cores derived from hESCs that were differentiatedin co-culture with OP9 cells for 3 days further grew into dispersedblast colonies with hematopoietic and endothelial potential.

bFGF is necessary and sufficient for the formation of both colonies fromhESCs. bFGF supported both mesenchymal and blast colony formation. Incontrast, in the absence of bFGF, neither VEGF, nor PDGF-BB (FIG. 1A),SCF, IGF1, or HGF (data not shown), alone or in combination, supportedformation of either colony. While PDGF-BB (10 ng/ml) alone did notsupport colony formation, PDGF-BB in combination with bFGF significantlyincreased the yield and size of mesenchymal colonies compared to bFGFalone (FIG. 1A). VEGF alone (20 ng/ml) did not support colony formationbut its addition to bFGF cultures slightly increased the number of blastcolonies, but inhibited formation of mesenchymal colonies (FIG. 1A).Cells that gave rise to each colony type constituted approximately 2-3%of total hESC-derived cells (FIG. 1B).

To determine if cells within the mesenchymal colonies can give rise tocells of the hematovascular lineage, individual mesenchymal colonieswere picked from the methylcellulose on day 5-7 and plated onto OP9cells in alpha-MEM medium with 10% FBS, and the cytokines SCF (50ng/ml), TPO (50 ng/ml), IL-3 (10 ng/ml), and IL-6 (20 ng/ml). After 4days of culture, cells were harvested and analyzed by flow cytometry orstained in situ with rabbit anti-human CD144 (VE-cadherin; 1 μg/ml;eBioscience, San Diego, Calif.) in combination with mouse anti-humanCD43 (0.5 μg/ml; BD Bioscience) or mouse anti-human Calponin (0.5 μg/ml;Thermo Fisher Scientific) primary antibodies, followed by a mixture ofsecondary cross-absorbed donkey anti-mouse IgG-DyLight 594 and donkeyanti-rabbit IgG-DyLight-488 (both at 2 μg/ml; Jackson ImmunoResearchLaboratories, Inc., West Grove, Pa.) antibodies.

The mesenchymal colonies originated from precursors that gave rise toendothelial and mesenchymal cells, i.e. mesangioblasts. As explained inExample 1, MSCs expanded from mesenchymal colonies in adherent culturesdid not give rise to hematopoietic or endothelial cells when coculturedwith OP9 cells. In contrast, approximately 70% of mesenchymal coloniesisolated from day 5-7 colony-forming cultures in semisolid media gaverise to CD31⁺CDl44(VE-cadherin)⁺ endothelial cells when cocultured withOP9 cells. (FIG. 1D and E, upper panels). The mesenchymal colonies,therefore, originated from common precursors for endothelial andmesenchymal lineages, i.e., mesangioblasts. In contrast, blast coloniescontained CD31⁺CD43⁺ hematopoietic cells and could give rise toendothelial cells (FIG. 1D and E lower panels).

The endothelial potential of mesenchymal colonies could be significantlyenhanced with the addition of bone morphogenic protein 4 (BMP4) to theclonogenic assay medium (3.2±2.4% CD31⁺CD43⁻ cells without BMP4 vs.11.6±0.5 with 5 ng/ml BMP4).

Example 3 Generating and Isolating a Population of Cells SubstantiallyEnriched in Lateral Plate/Extraembryonic Mesoderm Cells

Genetic profiling of H1 hESCs differentiated in OP9 coculturesdemonstrated selective commitment toward mesodermal and endodermallineages with no detectable ectoderm (tropho-, neuro-, or surfaceectoderm) (FIG. 2). The cells became committed to mesendoderm by day 2of culture, when synchronous expression of primitive streak genes(MIXL1, T, and EOMES) was detected. At subsequent days of culture,mesoderm- and endoderm-specific genes and, eventually, endoderm- andmesoderm derivative-specific genes were expressed. Of the mesodermalgenes, those characteristic of the lateral plate/extraembryonicmesodermal subset (FOXF1, HAND1, NKX2-5, GATA2) were expressedconsistently, while expression of genes of the axial (CHRD, SHH),paraxial (MEOX1, TCF15), or intermediate (PAX2, PAX8) subsets was notconsistent. Apelin receptor (APLNR) expression is strongly induced andup-regulated on days 2-3 of differentiation, concurrently withmesodermal commitment.

To characterize APLNR expression and the cells that express it, hESCsdifferentiated in OP9 co-cultures were stained with monoclonalantibodies specific for Apelin receptor (APLNR) (R&D Systems) incombination with antibodies against CD30, KDR, PDGFRA, T, and FOXA2.Undifferentiated hESCs and hESC-derived cells on day 1 of OP9 co-culturewere APLNR negative (FIG. 3A, Day 1 panels). Expression of APLNR wasstrongly up-regulated in cells co-cultured with OP9 cells for 2-3 days(FIG. 3A, Day 2 and 3). On day 2, 15-20% of cells were APLNR⁺ and by day3, 60-70% of cells were APLNR⁺. This upregulation coincided withmesodermal commitment, as evidenced by the upregulation of mesodermalmarkers, such as KDR (VEGFR2), T, and PDGFRA (FIG. 3A, Day 2 and 3panels). The number of APLNR⁺ cells gradually decreased on subsequentdays (FIG. 3B). Conversely, hESC markers (e.g. CD30) were successivelydown-regulated,

While PDGFRA is expressed only at low levels in day 2 co-cultures, APLNRis expressed at high density as early as day 2 of co-culture allowingseparation of APLNR positive from APLNR negative cells. On days 2, 2.5,and 3 of H1/OP9 cell co-culture, APLNR⁺ and APLNR⁻ cells were separatedby magnetic sorting and gene expression was analyzed by microarrayanalysis.

MIXL1, T, and EOMES, indicative of primitive streak cells (mesendoderm),were all expressed in APLNR⁺ cells, while transcripts associated withneural crest/neuroectoderm (POU4Fl, SOX1, SOX2, SOX3, SOX10) could notbe detected (FIG. 4A and 4B). As expected, APLNR⁺ cells were enriched inTCF21 mesoderm-specific transcripts, whereas transcripts markingpan-endoderm (FOXA2, APOA1), definitive (FOXA1, TMPRSS2), and visceral(TTR, AFP) endoderm were found in APLNR⁻ cells (FIG. 4A and C).

Interestingly, APLNR⁺ cells expressed FOXF1, IRX3, BMP4, WNT5A, NKK2.5,HAND1, and HAND2 representative of lateral plate/extraembryonicmesoderm, but not markers of paraxial/myogenic (MEOX1, TCF15, PAX3,PAX7) and intermediate (PAX2, PAX8) mesoderm in the embryo. This dataindicates that rather than being a total population of cells committedto mesendodermal development, APLNR⁺ cells represent mesoderm, or likelyits subpopulation reminiscent of lateral plate/extraembryonic mesoderm(FIG. 3C and FIG. 4).

To further confirm mesodermal identity, APLNR⁺ cells were analyzed forexpression of T, a marker of early mesoderm, and FOXA2, a marker ofendoderm. As shown in FIG. 3A, APLNR⁺ cells are T⁺ and maintain Texpression until it subsides on day 4. In contrast, FOXA2⁺ cells, whichcomprised less than 5% of total cells in culture, did not express APLNR.Thus, APLNR⁺ cells are T⁺FOXA2⁻ mesodermal precursors on day 2-3 ofculture.

To further support the notion that APLNR⁺ cells are mesodermalprecursors, H1/OP9 cell co-cultures were supplemented with inhibitors ofmesoderm formation SB431542 (5 μg/ml) or DKK1 (150 μg/ml). APLNR⁺ cellscould not be detected in cultures that received the inhibitors ofmesoderm formation (FIG. 3B), confirming that APLNR⁺ cells aremesodermal. Further, mesenchymal and blast colony-forming potential wasfound exclusively within the APLNR⁺ cell population (FIG. 3D), furtherconfirming that both mesangiogenic mesenchymal and hemangiogenic blastcolonies are formed by APLNR⁺ mesodermal precursors.

Example 4 Enrichment of Mesangioblasts Derived from hESCs UnderSerum-Free Conditions Through Isolation of APLNR⁺ LateralPlate/Extraembryonic Mesoderm Cells

To identify the origin of mesenchymal colonies and obtain a populationof cells enriched in mesangioblasts, pluripotent stem cells wereco-cultured with OP9 for 2-3 days to induce mesoderm formation. Afterdepletion of OP9 cells with mouse-specific CD29 antibodies, APLNR⁺ andAPLNR⁻ cells were isolated using magnetic sorting. Colony formationassays in semisolid media in presence of bFGF demonstrated thatmesangioblast and hemangioblast potential was confined solely to theAPLNR⁺ fraction (FIG. 3D). Approximately 1 to 5% of cells within APLNR⁺fraction possessed mesangioblast activity.

The invention has been described in connection with what are presentlyconsidered to be the most practical and preferred embodiments. However,the present invention has been presented by way of illustration and isnot intended to be limited to the disclosed embodiments. Accordingly,those skilled in the art will realize that the invention is intended toencompass all modifications and alternative arrangements within thespirit and scope of the invention as set forth in the appended claims.

1. A method of generating a clonal population of primate mesenchymalstem cells, the method comprising the steps of: culturing aheterogeneous, single-cell suspension of primate cells that containsmesenchymal progenitors in a serum-free, semi-solid medium containingbetween about 5 and about 100 ng/ml bFGF until independent coloniesform; and culturing one of the independent colonies in a serum-free,liquid medium containing between about 5 and about 100 ng/ml bFGF toobtain a substantially pure clonal population of MSCs.
 2. The method ofclaim 1, wherein the heterogeneous suspension is obtained in a methodcomprising the steps of: co-culturing pluripotent primate cells withbone marrow stromal cells in a medium that supports differentiation forbetween two and five days until differentiated cells are formed; andsuspending the differentiated cells.
 3. The method of claim 2, whereinthe pluripotent cells are selected from the group consisting ofembryonic stem cells (ESCs) and induced pluripotent stem (iPS) cells. 4.The method of claim 2, further comprising the step of depleting cellsnot derived by in vitro differentiation of pluripotent cells from theheterogeneous suspension.
 5. The method of claim 4, wherein thedepleting step comprises the steps of: non-covalently binding the cellsto be depleted to paramagnetic monoclonal antibodies specific forepitopes on the cells to be depleted; and segregating the antibody-boundcells with a magnet.
 6. The method of claim 2, wherein the bone marrowstromal cells are mouse OP9 cells.
 7. The method of claim 1, wherein theheterogeneous suspension is obtained in a method comprising the stepsof: dissociating an embryoid body to single cells; and suspending thesingle cells.
 8. The method of claim 1, wherein the single-cellsuspension is cultured for between ten to twenty days.
 9. The method ofclaim 1, wherein the semi-solid medium contains between about 20 andabout 100 ng/ml bFGF.
 10. The method of claim 1, wherein the semi-solidmedium contains about 5 ng/ml bFGF.
 11. The method of claim 1, whereinthe semi-solid medium contains about 1% methylcellulose.
 12. The methodof claim 1, wherein the semi-solid medium contains between about 10ng/ml and about 20 ng/ml PDGF-BB.
 13. The method of claim 1, whereincells in the single-cell suspension express MIXL1 and T.
 14. The methodof claim 1, wherein the ptimate cells are of human origin.
 15. Themethod of claim 1, wherein the MSCs are cultured in the presence of anextracellular matrix protein.
 16. The method of claim 15, wherein theextracellular matrix protein is selected from the group consisting ofMatrigel®, collagen, gelatin and fibronectin.
 17. The method of claim 1,wherein the mesenchymal colonies express FOXF1, MSX1, MSX2, SNAI1,SNAI2, SOX9 and RUNX2.
 18. The method of claim 1, wherein themesenchymal colonies express CD44, CD56, CD140a, CD146, and CD105, butdo not express CD31, CD43, CD45 and VE-cadherin.
 19. The method of claim1, wherein the method comprises the step of: observing at least onemesenchymal characteristic of colonies formed during culture in theserum-free, semi-solid medium, thereby confirming identification ofmesenchymal progenitors in the suspension.
 20. The method of claim 19wherein the at least one mesenchymal characteristic is selected from thegroup consisting of a functional characteristic, a morphologicalcharacteristic and a phenotypical characteristic.
 21. The method ofclaim 20, wherein the functional characteristic is selected from thegroup consisting of (1) growth stimulation by factors that promotemesenchymal cell growth (e.g., PDGF-BB, EGF and TGF-alpha) and growthsuppression by factors involved in mesodermal differentiation (e.g.,VEGF, TGF-beta and Activin A) and (2) differentiation into osteogenic,chondrogenic or adipogenic cell lineages.
 22. The method of claim 20,wherein the morphological characteristic is selected from the groupconsisting of (1) a tightly packed, round-shaped cell aggregatemeasuring 100-500 μm in diameter; and (2) lack of dense outer cell layerand irregular inner structure.
 23. The method of claim 20, wherein thephenotypical characteristic is selected from the group consisting of (1)expression of CD44, CD56, CD105, CD146, and CD140a, but no expression ofCD31, CD43, CD45 and VE-cadherin, (2) expression of FOXF1, MSX1, MSX2,SNAI1, SNAI2, SOX9 and RUNX2 and (3) expression of vimentin, alphasmooth muscle actin, and desmin.
 24. The method of claim 19, wherein themethod further comprises counting colonies to estimate a number ofmesenchymal progenitors in the heterogeneous suspension.
 25. A cellpopulation comprising: a substantially pure line of clonally-derivedmesenchymal stem cells positive at least for CD44, CD56, CD73, CD146,CD140a and CD105, but negative for CD31, CD43, CD45 and VE cadherin. 26.The cell population of claim 25, wherein the population comprises atleast 99% mesenchymal stem cells.
 27. A method of generating apopulation of primate Apelin receptor-positive lateral plate mesodermcells, the method comprising the steps of; culturing primate pluripotentstem cells in a medium that supports differentiation until Apelinreceptor is expressed isolating Apelin receptor-positive lateral platemesoderm cells.
 28. The method of claim 27, wherein the primatepluripotent stem cells are co-cultured with bone marrow stromal cellsfor about two to about five days.
 29. The method of claim 27 whereinabout 1% to about 5% of the Apelin receptor-positive cells havehemangioblast and mesangioblast potential.